Toxicological and pharmacological evaluation of Discaria americana Gillies & Hook ( Rhamnaceae ) in mice

Medicinal plants (e.g. Discaria americana) have been used by populations for centuries. However, popular use is not enough to validate these plants as safe and effective medicinal products. The present study sought to evaluate the acute and subacute toxicity as well as the anxiolytic and antinociceptive effects of D. americana root bark and aerial parts extracts in mice. In acute toxicity studies, mice were treated with single intraperitoneal doses of the aforementioned extracts. Subacute toxicity studies were performed by oral administration of the extracts over 14 days. Anxiolytic studies consisted of the elevated plus maze method, and antinociceptive studies were based on the hot plate test. The LD50 value for D. americana aerial parts extract was established at >500 mg/kg, and for the root bark extract, 400 mg/kg. D. americana aerial parts extract produced anxiolytic (250 mg/kg) and antinociceptive effects (125, 200 and 250 mg/kg). Conversely, D. americana root bark extract showed neither anxiolytic nor antinociceptive effects in mice.


INTRODUCTION
Medicinal plants have been used by populations for centuries.Even now, many plants play a key role in world health (Calixto, 2000).However, popular use is not enough to validate these plants as safe and effective medicines (Agra et al., 2007).Toxicity studies are required to evaluate the toxicity levels and adverse effects associated with medicinal plants.In addition to toxicity studies, study of the pharmacological activity of these plants is also important.
Discaria americana Gillies & Hook is a shrub from the Rhamnaceae family.This plant is known by the common names "Quina-do-Brasil" and "Quina-do-Rio-Grande" (Brazil) or "Coronillo del campo" and "Quina del campo" (Uruguay) (Correa, 1984).D. Americana root bark is used by local populations against stomach and skin diseases, diabetes, fever (antipyretic), and as a tonic (Záchia, Moraes, 1999).However, almost no studies have assessed the toxicological and pharmacological properties of this plant; only one report, describing strong antioxidant activity of the essential oil extracted from the aerial parts of D. americana (Rodriguez, Murray, 2008), was located by a review of the literature.
Cyclopeptide alkaloids and cyclopeptides (Giacomelli et al., 2004;Giacomelli, 2005) have also been isolated from D. americana.However, almost nothing has been reported on the toxic and pharmacological effects of these compounds.Among the alkaloids, frangulanine has a sedative effect, whereas discarine A and discarine B reportedly show antibacterial activity (Maldaner, 2005).Some Rhamnaceae plants have been reported to show high levels of toxicity and adverse effects.Liver and lung damage and death have been observed in mice given Karwinskia humboldtiana (Bermudez et al., 1986).This plant also caused a decrease in ATP levels in the kidney and blood (Jaramillo-Juárez et al., 2005) and central nervous system damage in rats (Becerra-Verdin et al., 2009).Rhamnus cathartica, another plant from the same family, is known to be hepatotoxic in mice (Lichtensteiger et al., 1997).Therefore, according to the toxicity shown by other Rhamnaceae, evaluation of the toxicity of D. americana is paramount.
Thus, the present study sought to evaluate the acute and subacute toxicity of D. americana aerial parts and root bark extracts, as well as the potential anxiolytic and antinociceptive effects of these extracts, in mice.

Preparation and identification of D. americana extracts
Dried ground root bark (1080 g) and aerial parts (1200 g) of D. americana were extracted with methanol (MeOH) (6 L) in a Soxhlet apparatus for 12 h.The resulting MeOH extracts were filtered and concentrated under vacuum, yielding the crude residues of the plant root bark and aerial parts (2.79 and 0.69% yield, respectively).The extracts were dissolved in dimethyl sulfoxide (DMSO) for administration in mice.
Four cyclopeptides were isolated from the root bark of D. americana (discarene C, discarene D, discarine M and discarine N), as well as nine cyclopeptide alkaloids (adoutine Y, adoutine Y′, franganine, frangulanine, discarine A, discarine B, discarine C, discarine D and myrianthine A), betulinic acid, and three steroids (b-sitosterol, b-sitosterol-3-O-6-b-D-glucopyranoside and b-sitosterol-3-O-6-stearoyl-b-D-glucopyranoside).The steroids b-sitosterol and b-sitosterol-3-O-6-b-D-glucopyranoside, three pentacyclic triterpenes (betulinic acid, ursolic acid and ceanotic acid) and an ester derived from ferulic acid were isolated from the plant aerial parts.The isolated compounds were identified by direct comparisons with authentic samples by thin layer chromatography (TLC) and on the basis of NMR spectral data.TLC was performed on precoated silica gel 60 F 254 plates (Merck ® ) (mobile phase: mixture of CHCl 3 and MeOH) and detection was achieved by UV light (254 nm), by spraying with Dragendorff's reagent, and by spraying with 10 % H 2 SO 4 , followed by heating (Giacomelli et al., 2004;Giacomelli, 2005).cilities, were kept in an air-conditioned room (22 ± 2 o C), under a 12-hour light/dark cycle (lights on at 8:00 a.m.), with water and food (Nuvital ® chow, PR, Brazil) ad libitum.A total sample of 270 mice was used for toxicity and pharmacology studies.For each test, animals were divided into groups of 10 animals.The experiments were carried out with the approval of the Universidade Regional Integrada do Alto Uruguai e das Missões Research Ethics Committee (protocol no.023/07).

Acute toxicity studies
Groups of 10 mice were injected with single doses of D. americana root bark extract (50, 100, 300, 400 and 500 mg/kg), plant aerial parts extract (500 mg/kg) and DMSO (control).The dose of 500 mg/kg was established as the highest dose of the plant extract for determination of LD 50 .Mice were injected intraperitoneally, as this route of administration is associated with rapid absorption, thus enabling evaluation of exposure during a short period of time.
Signs of general toxic effects, such as weight loss, seizures, or death, were observed continuously for 2 h and then monitored over a 48-hour period.After 48 h, animals were euthanized and necropsied for gross observation of the heart, kidneys and liver, as well as blood sample collection.The study was carried out as by Maciel et al. (2000), with the exception of exposure time (48 hours).

Subacute toxicity studies
D. americana root bark extract (50 and 100 mg/kg) and aerial parts extract (300 and 400 mg/kg) were administered to groups of 10 mice by once-daily gavage for 14 days.Control group animals received the vehicle (DMSO) under the same conditions.Toxic manifestations, such as seizures, weight loss, and death, were monitored daily.At the end of the 14-day period, animals were euthanized and necropsied for macroscopic observation of the heart, kidneys and liver, as well as blood sample collection.Again, the study was carried out as by Maciel et al. (2000), except for the route of administration.

Blood analysis
Blood was collected in heparinized tubes and used immediately.Tubes were centrifuged at 3000 rpm for 10 min to obtain serum, which was then analyzed.Transaminase (AST and ALT) activity was determined enzymatically as described by Bergmeyer (1978), using commercial kits from Labtest Diagnóstica S. A ® in a semiautomatic LABQUEST ® biochemical analyzer.

Evaluation of anxiolytic effect
The elevated plus maze (EPM) method was used to assess the anxiety level of mice.The maze consists of two opposed open arms and two opposed enclosed arms, bounded by 30-cm high walls.Each arm measures 50 x 10 cm, with a shared central area of 10 x 10 cm.The maze is kept 50 cm above the floor.
One hour after treatment, mice were placed on the central area of the maze.The number of entries into the enclosed and open arms was recorded, as was the time spent in each arm.Time spent in the open arms was considered as the measure of anxiolytic effect (Pellow et al., 1985).

Evaluation of antinociceptive effect
The experiment started 60 min (time zero) after intraperitoneal administration of D. americana root bark extract (50, 100 and 200 mg/kg) and aerial parts extract (125, 200 and 250 mg/kg) to groups of 10 mice.Animals were placed individually on a hot plate kept at a constant temperature of 55 °C.The time to first paw lick (henceforth "latency") was recorded.Measurements were performed at 0, 15, 30 and 60 min after the first thermal stimulus.To avoid damage to the animals' paws, the maximum time spent on the plate was 30 s.Therefore, a latency period of 30 s was defined as complete antinociception (Ankier, 1974).For comparison, the same experiment was conducted with paracetamol 400 mg/kg (positive control) and DMSO (control).

Acute toxicity
To assess acute toxicity of D. americana, mice were injected with aerial parts and root bark extracts and with DMSO (control).During the experiment, the occurrence of adverse affects and the mortality of mice were observed.The maximum dose of 500 mg/kg of D. americana aerial parts extract did not cause death in mice; hence, the LD 50 value was established as >500 mg/kg (Table I).
According to Saraswat et al. (1996), ursolic acid, a triterpene constituent of D. americana, has hepatoprotective activity.However, acute administration of the plant aerial parts extract at the dose of 500 mg/kg caused a significant increase in transaminase (ALT and AST) levels (Table II).High serum levels of these enzymes are a marker of injury, usually liver damage.Furthermore, autopsy of these animals revealed a variable number (1 to 4) of opalescent oval lesions, approximately 0.5 cm in diameter, on the liver.These hepatic lesions were observed in 70% of animals.
Acute administration of D. americana root bark extract caused mortality proportional to the administered doses.At the highest dose level (500 mg/kg), the mortality rate was 60%.The LD 50 was established as 400 mg/kg.At this dose, there was a significant increase in AST levels.
At 300 mg/kg, 40% of mice died, and half of those that survived showed evidence of liver injury and significantly increased ALT levels.At the dose of 50 and 100 mg/kg, there was no mortality, but ALT levels were increased as compared to mice in the control group.
The weight of mice that survived acute exposure was evaluated 48 h after administration of the extracts.There was no significant weight loss in mice treated with D. americana extracts as compared to the control group.Furthermore, there was no significant difference in liver weight/body weight of mice treated with D. americana in relation to the control group (Table III).
No macroscopic changes were observed in the kidneys and hearts of mice treated with D. americana extracts.Furthermore, there was no significant difference in kidney weight or heart weight/body weight of mice treated with D. americana in relation to the control group.No seizure activity was observed in acute toxicity studies.There were no deaths or adverse effects in the control group.

Subacute toxicity
For assessment of subacute toxicity, D. americana extracts and DMSO were orally administered to mice for   14 days.General toxic effects and mortality were observed during the treatment period.At a dose of 400 mg/kg, the aerial parts extract caused 20% mortality (Table I), moderate weight loss (Table III), and a significant increase in ALT and AST levels (Table IV).At 300 mg/kg, there were no deaths or significant weight loss, although ALT and AST levels did increase.
At a dose of 100 mg/kg, D. americana root bark extract led to significant weight loss and increased ALT and AST levels, as well as a 20% mortality rate.A dose of 50 mg/kg was associated with 10% mortality and increased serum AST levels, but no significant weight loss.
The greater toxicity of D. americana root bark extract as compared to the aerial parts extract can be attributed to the alkaloids present in the root bark.A review of the literature showed that some of these alkaloids have known toxic effects in mice (Tabosa et al., 2006).Furthermore, some alkaloids are toxic -mainly hepatotoxi -in humans (Bolzan et al., 2007).
No macroscopic changes were observed in the liver, kidneys and heart of mice treated with D. americana extracts.There were no significant differences in the ratio of weight of these organs to body weight in mice treated with D. americana as compared to the control group.No seizure activity was observed in subacute toxicity studies.There were no deaths and no adverse effects in the control group.
Although important parameters were evaluated in these acute and subacute toxicity studies, further assessments are needed before intake of D. americana can be considered safe in humans.

Pharmacology
This study evaluated the anxiolytic effect of D. americana in mice using the elevated plus maze (EPM) method.The potential antinociceptive effect of the plant was evaluated with the hot plate test.No toxic doses or doses that showed lower toxicity effects were used in the pharmacology portion of this study.These doses were determined according to acute toxicity studies.For D. americana root bark extract, 50 mg/kg (1/8 LD 50 ), 100 mg/kg (1/4 LD 50 ) and 200 mg/kg (1/2 LD 50 ) doses were used.The aerial parts extract of D. americana showed a LD 50 of >500 mg/kg.At this dose, toxic manifestations were observed; therefore, half of this dose (250 mg/kg) was used for pharmacology studies.The 125 and 200 mg/kg doses were also used in pharmacological studies in order to evaluate the possibility of a dose-dependent effect.
In the anxiolytic studies, the control group spent most of the 10 min in the enclosed arms of the EPM, indicating anxiety.Data showed an increase in time spent in the open arms among mice treated with D. americana aerial parts extract at the dose of 250 mg/kg.This reduction in anxiety could be associated with ursolic acid, an important component of the aerial parts of D. americana that has known anxiolytic properties (Durst et al., 2002).A similar effect occurred in the positive control group (DZP 1.5 mg/kg).D. americana root bark extract had no anxiolytic effect (Table V).
Antinociceptive studies revealed that mice treated with D. americana aerial parts extract at a dose of 125 mg/kg had a significant reduction in nociceptive response at 0 and 15 s.The 200 mg/kg dose caused a significant increase in latency at all periods analyzed.At a 250 mg/kg dose, the nociceptive response was > 30 s.A trend toward dose-dependent increase in the analgesic effect of D. americana aerial parts extract was observed.This striking antinociceptive potential of D. americana  can be ascribed to three components that are present in the plant: the triterpenes ursolic acid (Tapondjou et al., 2003) and betulinic acid (Yogeeswari, Sriram, 2005) and the steroid b-sitosterol (Cechinel Filho, Yunes, 1998), which are known antinociceptive agents.Pretreatment of animals with paracetamol (400 mg/kg), the positive control used in this study, also caused a significant antinociceptive effect (nociceptive response >30 s).Conversely, D. americana root bark extract did not reduce the nociceptive response in mice (Table VI).
In conclusion, the present study showed that D. americana root bark extract produces toxic effects at low doses and was not successful as an anxiolytic or antinociceptive agent.Conversely, an aerial parts extract of the same plant showed low toxicity even when administered at the highest doses, and successfully reduced anxiety and nociception in mice.However, to protect human health, further toxicity studies are necessary if this plant is to be used as a herbal remedy.On the basis of the findings reported herein, D. americana can be considered a promising source for development of new anxiolytic and analgesic agents.

TABLE I -
Effect of acute and subacute administration of D. americana extracts on mortality in mice.N =10

TABLE II -
Effect of acute administration of D. americana extracts on serum transaminase levels (U/L) in mice

TABLE III -
Effect of acute and subacute D. americana exposure on weight and liver weight/body weight of mice

TABLE IV -
Effect of subacute administration of D. americana extracts on serum transaminase levels (U/L) in mice

TABLE V -
Time spent in open arms of the elevated plus maze after exposure to D. americana extracts Values are expressed as mean ± standard deviation.N=10.(*) Statistically different from controls, p<0.05 (ANOVA/ Tukey's test).